JP3895262B2 - Hepatitis C disease test method - Google Patents

Description

Jacalin処理のものを無処理のものと比べると、ＨＣＶ患者の凹みの周辺の白い沈澱物がJacalin処理により観察されなくなった。
この結果から、ＨＣＶ患者のクリオグロブリン沈澱物には変性ＩｇＭとGalβ1-3GPとの複合体が含まれるもの考えられる。しかし、Jacalinが複合体を形成するGalβ1-3GPと特異的に結合し、凹みの周辺に移動することが出来なくなり、凹みの周辺の白い沈澱物がJacalin処理により観察されなくなったと考えられる。
【００１８】
実施例１
この実施例では、ＨＣＶ患者のクリオグロブリン沈澱物に含まれる変性ＩｇＭとGalβ1-3GPとの複合体を測定することを目的とする。
Galβ1-3GPと親和性が確認されたJacalinをコーティングした酵素結合免疫収着検定法（以下「ＥＬＩＳＡ」という。）反応用プレートを以下の手順で作製した。
１．プレート（ｃｏｓｔａｒ社製６穴丸底 高吸差能タイプ）各穴にコーティングバッファーで稀釈したJacalin Pure（ＥＹ社製）溶液（１μｇ／ｍＬ）を１００μＬずつ添加し２〜８℃で一晩放置する。
２．上記プレートを洗浄液（９０ｍＭ リン酸緩衝液（ＮａＣｌ，８．０ｇ；Ｎａ_２ＨＰＯ_４，１．１５ｇ；ＫＨ_２ＰＯ_４，０．２ｇ；ＫＣｌ，０．２ｇ；Ｔｗｅｅｎ２０，０．５ｇ；ＤＷ，１Ｌ）、以下洗浄液は同様のものを使用した。）で３回洗浄し、イオン交換水で５倍に稀釈したブロッキング剤（日本油脂製Ｎ１０２ブロッキング剤）を各プレート穴へ１５０μＬ添加し、室温で２時間放置する。
３．上記プレートを洗浄液で３回洗浄し、水分を完全に除去する為２，５００ｒｐｍ、１０分間遠心する。プレートシーラーを貼り付けて冷蔵庫で４８時間以上静置して使用する。
【００１９】
次に、健常者１０名（表２のＮｏ．１〜１０）から血清を採取し、このプレートを用いて下記の手順でＥＬＩＳＡによる検定を行った。
１．反応プレートを室温に戻し、洗浄液で３回洗浄後タッピングして水分を除去する。
２．上記プレートに３７℃で保存した血清検体５０μｌを注入後４℃で２時間静置する。
３．冷洗浄液で３回洗浄後、抗体稀釈液（０．８５％ＮａＣｌ）で５，０００倍に稀釈したＨＲＰ標識抗ヒトＩｇＭウサギ抗体（ダコ社）１００μＬを各プレート穴へ添加し、ミキサーにかけ、４℃で１時間反応させる。
４．冷洗浄液で３回洗浄後、ＴＭＢＺ発色試薬（クエン酸１水和物，１２．８０ｇ／Ｌ；Ｎａ_２ＨＰＯ_４，１０．９０ｇ／Ｌ；Ｈ_２Ｏ_２，０．１５ｍｌ／Ｌ；１０ｍｇ／ｍｌＴＭＢＺ，１０ｍｌ／Ｌ）１００μＬを各プレート穴へ添加し、室温で３０分間反応させる。
５．反応停止用リン酸溶液（リン酸，３４ｍＬ；ＤＷ，５００ｍＬ）１００μＬを各プレート穴に加え、反応を終了する。
６．プレートリーダーにかけて、４５０ｎｍで測光し吸光度（Ａｂｓ）を求める。
【００２０】
実施例２
ＨＣＶ患者１５名（表２のＮｏ．１１〜２５）から採取した血清を用いて、実施例１と同様の試験を行った。
実施例１（Ｎｏ．１〜１０）と実施例２（Ｎｏ．１１〜２５）の結果を表２に示す。
なお、同時に同血清検体をＨＣＶ・ＥＩＡ ２ｎｄ「アボット」キットで検査し、サンプルをＨＣＶ関連抗原と反応させた後の吸光度をカットオフ値の吸光度で割った値（Ｃ−ＩＤＸ）を表２に示す。このキットはＨＣＶコア抗原とＨＣＶ関連抗原を含み、サンプル中のＨＣＶ関連抗体の有無を検査する。
【００２１】
【表２】

この表２から、ＨＣＶ患者の吸光度（Ｎｏ．１１〜２５）は健常者のもの（Ｎｏ．１〜１０）に比べて有意に低いことがわかる。
【００２２】
JacalinはＣ型肝炎患者の変性したＩｇＭとＧａｌβ１−３ＧＰとの複合体も健常者の正常ＩｇＭ・Ｇａｌβ１−３ＧＰ複合体も区別無く固相に結合する。図３に示すように、健常者の正常ＩｇＭ・Ｇａｌβ１−３ＧＰ複合体を担体に固定化したJacalinと結合させて検知することができる。
しかし、Ｃ型肝炎患者のＩｇＭは参考例１で明らかなように健常者のＩｇＭよりも分子量が数千大きく、健常者のものとは異なる変性ＩｇＭである。このような変性ＩｇＭからなる複合体は冷却すると凝集し、Jacalinと結合することができなくなってしまうものと考えられる。その結果、図４に示すように、ＥＬＩＳＡによる検定による数値が低くなるものと考えられる。即ち、この値は、ＨＣＶ感染（たぶんリンパ球への感染）に伴う変性ＩｇＭ産生を示すものであると考えられる。即ち、本発明の方法は、Ｃ型肝炎患者の変性ＩｇＭを見積もる手段を与えるものである。
【図面の簡単な説明】
【図１】健常者とＣ型肝炎の患者から得た６つのクリオ沈澱物のＰＡＧＥパターンを示す図である。レーン１は健常者のもの、レーン２〜６はＨＣＶ患者のものを示す。矢印はｍｕ鎖バンドを示す。ｍｕ−０は通常のもの、ｍｕ−１とｍｕ−２は異常のものを示す。
【図２】冷アガロースゲル上のＣ型肝炎の患者と健常者のクリオ沈澱物リングを示す図である。No.１とNo.４は健常者のもの、No.２とNo.３はＨＣＶ患者のもの、No.１とNo.２はJacalinで処理したもの、No.３とNo.４は無処理のものを示す。No.３の凹みの周辺にのみ白い沈澱物が観察された。
【図３】ＥＬＩＳＡ法により、健常者の正常ＩｇＭ・Ｇａｌβ１−３ＧＰ複合体を担体に固定化したJacalinと結合させて検知する方法(実施例１)の概略を示す図である。
【図４】ＥＬＩＳＡ法により、Ｃ型肝炎患者の変性ＩｇＭ・Ｇａｌβ１−３ＧＰ複合体を担体に固定化したJacalinと結合させて検知する方法(実施例２)の概略を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for testing for hepatitis C disease.
[0002]
[Prior art]
When infected with hepatitis C virus (HCV), there are problems such as being easily chronic and having a high probability of causing cirrhosis. For this reason, a test technique that can confirm in a short time whether or not hepatitis C virus is present in the blood of a patient suspected of being infected with hepatitis C virus is important. Furthermore, although hepatitis C virus is present in the free state and / or immune complex in the body of an HCV-infected patient, the hepatitis C virus is in any state (ie free state or immune complex). There is no appropriate method for knowing whether it exists in the state), and the completion of such an inspection method has been desired.
[0003]
On the other hand, cryoglobulin is a thermal protein that precipitates from serum at low temperatures and has been detected in a wide range of diseases such as many myeloma, lymphoproliferative diseases, autoimmune diseases and infectious diseases (for example, non-patented) Reference 1). There have been reports on the relationship between cryoglobulinemia and hepatitis C (see, for example, Non-Patent Documents 2 and 3), and the presence of HCV-RNA in cryoglobulin has been confirmed (eg, non-patent documents). (See Patent Document 4). However, confirmation of cryoglobulin by the conventional method requires a large amount of HCV-infected serum, and it was difficult to detect a very small amount of cryoglobulin precipitate.
[0004]
The inventors have already developed and reported a simple method for detecting cryoglobulin (see, for example, Non-Patent Document 5). This method is a gel diffusion method using a cooled agarose gel plate and can be performed with a small amount of sample of about 10 μl. The cryoglobulin precipitation ring obtained by this method is very clear, and is particularly effective for the analysis of cryoglobulin in hepatitis C. Furthermore, the present inventors have developed a method of using this free virus phase separation detection technique as a test technique for hepatitis C virus (Japanese Patent Application 2001-230946).
[0005]
[Non-Patent Document 1]
Ministry of Health and Welfare Emerging and Re-emerging Infectious Diseases Research Project “Prevention and Epidemiology of Non-A Non-B Hepatitis, Clinical Comprehensive Study of Non-A Non-B Hepatitis” 1999 Research Report, Shiro Iino “ Study of cryoglobulinemia "
[Non-Patent Document 2]
Casato M, et.al., Lancet 1991; 337; 1047-8
[Non-Patent Document 3]
Disdier P, et.al., Lancet 1991; 338: 1151-2
[Non-Patent Document 4]
Agnello V, et. Al., N Engl J Med 1992; 327: 1490-5
[Non-Patent Document 5]
Okazaki T, et.al., C1inica1 Chemistry 1998; 44: 1558-1559
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for examining hepatitis C virus in serum with a simple apparatus and less complicated operation, and further to identify an immune complex peculiar to a person infected with hepatitis C virus. It provides a means by which this can be estimated, i.e., an effective method to identify hepatitis C patients.
[0007]
[Means for Solving the Problems]
As a result of diligent research to solve such problems, the present inventors have found that IgM of hepatitis C patients is a modified IgM different from that of healthy individuals, and that IgM binds to Galβ1-3GP. Formation of a complex, binding of this complex to a specific lectin, and aggregation of the complex of modified IgM and Galβ1-3GP at low temperature, that is, a cryoglobulin is obtained. It was. Based on these findings, the present inventors have completed a method for estimating an immune complex composed of abnormal IgM unique to a person infected with hepatitis C virus and a method for identifying hepatitis C patients. In addition, Galβ1-3GP is a glycoprotein having a structure of Galβ1-3GalNac present in the subject serum sample at the sugar chain end.
That is, the present invention comprises a step in which a subject's serum specimen is contacted with a carrier on which a lectin having an affinity for Galβ1-3GP is immobilized in a non-freezing cold state of less than 37 ° C. (freezing at less than 37 ° C.). In the cold state, after the carrier obtained in the previous step is cold-washed, the carrier is then contacted with an enzyme-labeled second antibody against human IgM (second step), and the carrier obtained in the previous step is cold-washed This is a method for examining hepatitis C, comprising the step of detecting a label bound to the carrier (third step).
[0008]
DETAILED DESCRIPTION OF THE INVENTION
As described above, the inventors have found that IgM of patients with hepatitis C is denatured IgM whose molecular weight is several thousand greater than that of normal individuals. The results are shown in Reference Example 1 described later.
Although IgM and Galβ1-3GP form a complex, it is considered that the complex of healthy subject IgM and Galβ1-3GP does not aggregate even at low temperatures. However, it is considered that the complex of denatured IgM and Galβ1-3GP from hepatitis C patients aggregates at low temperature, and this is cryoglobulin. It has already been reported that this aggregated state was observed on an agar gel (Non-patent Document 5).
[0009]
On the other hand, the present inventors have found that a complex composed of IgM and Galβ1-3GP specifically binds to a specific lectin. The results are shown in Reference Example 2 described later. A lectin is a protein having a sugar chain structure extracted from animals and plants, and causes a phenomenon similar to an agglutination / precipitation reaction or other specific antibody reaction. As a lectin having an affinity for Galβ1-3GP, for example, Jacalin which is a coconut lectin demonstrated in Examples is preferable. The affinity with Galβ1-3GP can be known, for example, by the method described in Reference Example 2 described later.
[0010]
A lectin (Jacalin) binds to a solid phase regardless of whether a complex of modified IgM and Galβ1-3GP of a patient with hepatitis C or a normal IgM · Galβ1-3GP complex of a healthy person.
However, cryoglobulin, which is a complex of modified IgM of Hepatitis C patients and Galβ1-3GP, aggregates at a low temperature, so that it cannot bind even if a lectin (Jacalin) is placed in the system as in the method of the present invention. It is thought that it will end up. Therefore, according to the method of the present invention, it is possible to estimate the amount of a complex composed of modified IgM and Galβ1-3GP that cannot bind to lectin (Jacalin).
[0011]
The first stage and the second stage of the test method for hepatitis C virus of the present invention are carried out in a freezing cold state of less than 37 ° C, preferably 1 to 10 ° C, more preferably around 4 ° C. At the same temperature.
The cold washing is performed at a temperature of less than 37 ° C., preferably 1 to 10 ° C., and usually using a 90 mM phosphate buffer or the like.
Further, the third stage does not need to be a low temperature and is usually performed at 37 ° C.
The enzyme-labeled second antibody is not particularly limited as long as it is an antibody against human IgM, but an HRP-labeled human IgM rabbit antibody or the like is generally used.
[0012]
【Example】
The following examples illustrate the invention, but are not intended to limit the invention.
Reference example 1
Analysis of cryoglobulin contained in the precipitate ring on the gel was performed using polyacrylamide gel electrophoresis (PAGE). First, the cryoprecipitate ring is cut out at a low temperature, and the liglobulin contained in the agarose gel is dissolved at 37 ° C. in Dulbecco phosphate buffer (pH 7) containing glycine. This lysate was treated with Tris-SDS-βME seprazol (Daiichi Kagaku Co., Ltd.), and 0.015 M Tris-0.195 M glycine buffer (pH 8.6) was used at a constant current of 40 mA and a 5-15% polyacrylamide gradient. Gel electrophoresis was performed for 60 minutes. After electrophoresis, the fractionated immunoglobulin was transferred to an Immobilon PVDF transfer membrane (Daiichi Kagaku) for 1 hour, and this membrane was blocked with skim milk (Block Ace, Dainippon Pharmaceutical). This was reacted with an anti-μ chain antibody (Dako A / S, Glostrup), and then colored with a POD-labeled rabbit antibody (Dako A / S, Glostrup). The result is shown in FIG.
Lane 1 is for healthy subjects and lanes 2-6 are for HCV patients. Among the HCV patients, abnormal mu fractions several kilodaltons larger than those in healthy subjects were confirmed for those with a large amount of cryoglobulin precipitation.
That is, from this result, it was confirmed that abnormal IgM was produced in patients with hepatitis C. This is thought to be evidence that HCV enters lymphocytes and is detrimental to the immune mechanism.
[0013]
Reference example 2
The composition A and the composition B were added to 750 mL of water, squeezed sufficiently, and then dissolved in a boiling water bath.
(1) Composition A:
NaCl 800mg
KCl 200mg
Sodium monohydrogen phosphate 1150mg
Sodium dihydrogen phosphate 200mg
(2) Composition B:
Sodium azide 0.75g
Glycine 56.25g
Agarose 4.50g
Upon dissolution, the mixture was stirred with a stirrer every 5 minutes to obtain a uniform agarose solution.
[0014]
Immediately after dissolution of the agarose solution, it is poured onto a 30 mm diameter plate and allowed to solidify at room temperature to obtain an agarose gel plate. About 7 recesses (wells) having a diameter of 5 mm were formed on this agarose gel plate, and these were stored refrigerated.
A serum sample (20 μL) collected from a hepatitis C virus infected person and a healthy person by a predetermined means was maintained at 37 ° C., and this was injected into a recess of an agarose gel plate at room temperature. Thereafter, the sample was allowed to stand for 20 minutes, and the serum sample was soaked into the gel plate.
[0015]
The gel plate was placed near 4 ° C. and incubated for 12 hours or longer. The results are shown in No. 3 (infected with hepatitis C virus) and No. 4 (healthy person) in FIG. Only in No. 3, a white precipitate ring was observed around the dent. That is, as in the previous report (Non-Patent Document 5), it is considered that the complex of modified IgM and Galβ1-3GP aggregated and precipitated around the recess.
[0016]
On the other hand, Jacalin Pure (manufactured by EY) was added to the above serum sample so that the concentration was 1 μg per 10 μl of the serum sample, allowed to stand for about 2 hours, and then injected into the dent of the plate of Reference Example 2, followed by the same operation as above. Went. The results are shown in No. 2 (infected with hepatitis C virus) and No. 1 (healthy person) in FIG. In any case, no white precipitate ring was observed around the recess.
These results are summarized in Table 1. In the table, “+” indicates that a white precipitate was observed around the dent, and “−” indicates that no white precipitate was observed.
[0017]
[Table 1]

Compared with the untreated one with the Jacalin treatment, the white precipitate around the dent of the HCV patient was not observed by the Jacalin treatment.
From this result, it is considered that the cryoglobulin precipitate of HCV patients contains a complex of modified IgM and Galβ1-3GP. However, it is considered that Jacalin specifically binds to Galβ1-3GP forming a complex and cannot move around the depression, and the white precipitate around the depression is not observed by the Jacalin treatment.
[0018]
Example 1
The purpose of this example is to measure the complex of modified IgM and Galβ1-3GP contained in cryoglobulin precipitates of HCV patients.
An enzyme-linked immunosorption assay (hereinafter referred to as “ELISA”) reaction plate coated with Jacalin, which was confirmed to have affinity for Galβ1-3GP, was prepared by the following procedure.
1. Plate (Costar 6-hole round bottom high-absorption type) Add 100 μL of Jacalin Pure (EY) solution (1 μg / mL) diluted with coating buffer to each hole and leave at 2-8 ° C. overnight. .
2. The plate was washed (90 mM phosphate buffer (NaCl, 8.0 g; Na ₂ HPO ₄ , 1.15 g; KH ₂ PO ₄ , 0.2 g; KCl, 0.2 g; Tween 20, 0.5 g; DW, 1 L). In the following, the same cleaning solution was used 3), and 150 μL of blocking agent (N102 blocking agent made by NOF Corporation) diluted 5 times with ion-exchanged water was added to each plate hole. Leave for hours.
3. The plate is washed 3 times with a washing solution and centrifuged at 2500 rpm for 10 minutes to completely remove moisture. Apply plate sealer and leave it in the refrigerator for 48 hours or more.
[0019]
Next, serum was collected from 10 healthy subjects (No. 1 to 10 in Table 2), and assayed by ELISA using the following procedure using this plate.
1. The reaction plate is returned to room temperature, washed three times with a washing solution and tapped to remove moisture.
2. After injecting 50 μl of serum specimen stored at 37 ° C. onto the plate, the plate is allowed to stand at 4 ° C. for 2 hours.
3. After washing 3 times with a cold washing solution, 100 μL of HRP-labeled anti-human IgM rabbit antibody (Dako) diluted 5,000 times with antibody dilution solution (0.85% NaCl) was added to each plate hole, applied to a mixer, 4 React at 1 ° C. for 1 hour.
4). After washing three times with a cold washing solution, the TMBZ coloring reagent (citric acid monohydrate, 12.80 g / L; Na ₂ HPO ₄ , 10.90 g / L; H ₂ O ₂ , 0.15 ml / L; 10 mg / ml TMBZ) , 10 ml / L) is added to each plate hole and allowed to react for 30 minutes at room temperature.
5). 100 μL of a reaction stopping phosphoric acid solution (phosphoric acid, 34 mL; DW, 500 mL) is added to each plate hole to complete the reaction.
6). Using a plate reader, photometry is performed at 450 nm to determine the absorbance (Abs).
[0020]
Example 2
The test similar to Example 1 was done using the serum extract | collected from 15 HCV patients (No. 11-25 of Table 2).
Table 2 shows the results of Example 1 (No. 1 to 10) and Example 2 (No. 11 to 25).
At the same time, the same serum specimen was examined with the HCV · EIA 2nd “Abbott” kit, and the absorbance (C-IDX) obtained by reacting the sample with the HCV-related antigen divided by the absorbance of the cutoff value (C-IDX) is shown in Table 2. Show. This kit contains an HCV core antigen and an HCV-related antigen and tests for the presence of HCV-related antibodies in the sample.
[0021]
[Table 2]

From Table 2, it can be seen that the absorbance (No. 11-25) of HCV patients is significantly lower than that of healthy individuals (No. 1-10).
[0022]
Jacalin binds to a solid phase regardless of whether a complex of modified IgM and Galβ1-3GP of a patient with hepatitis C or a normal IgM · Galβ1-3GP complex of a healthy person. As shown in FIG. 3, the normal IgM · Galβ1-3GP complex of a healthy person can be detected by binding to Jacalin immobilized on a carrier.
However, IgM of a hepatitis C patient is a modified IgM having a molecular weight several thousand larger than that of a healthy person as apparent from Reference Example 1, and different from that of a healthy person. It is considered that a complex composed of such modified IgM aggregates when cooled and cannot bind to Jacalin. As a result, as shown in FIG. 4, it is considered that the numerical value obtained by the ELISA test is lowered. That is, this value is considered to indicate degenerate IgM production associated with HCV infection (perhaps infection of lymphocytes). That is, the method of the present invention provides a means for estimating degenerative IgM in hepatitis C patients.
[Brief description of the drawings]
FIG. 1 shows PAGE patterns of six cryoprecipitates obtained from healthy subjects and hepatitis C patients. Lane 1 shows that of a healthy person, and lanes 2 to 6 show those of an HCV patient. The arrow indicates the mu chain band. mu-0 is normal, and mu-1 and mu-2 are abnormal.
FIG. 2 shows the cryoprecipitate ring of hepatitis C patients and healthy individuals on a cold agarose gel. No.1 and No.4 are for healthy subjects, No.2 and No.3 are for HCV patients, No.1 and No.2 are treated with Jacalin, No.3 and No.4 are untreated The thing is shown. A white precipitate was observed only around the recess of No. 3.
FIG. 3 is a diagram showing an outline of a method (Example 1) in which normal IgM · Galβ1-3GP complex of a healthy subject is detected by binding to Jacalin immobilized on a carrier by ELISA.
FIG. 4 is a diagram showing an outline of a method (Example 2) in which a denatured IgM · Galβ1-3GP complex of a hepatitis C patient is bound to Jacalin immobilized on a carrier and detected by ELISA.

Claims (4)

A step in which a serum sample of a subject is brought into contact with a carrier on which a lectin having an affinity for Galβ1-3GP is immobilized in a freezing cold state of less than 37 ° C., a carrier obtained in the previous step in a freezing cold state of less than 37 ° C. C type comprising: a step of contacting the carrier with an enzyme-labeled second antibody against human IgM after cold washing; and a step of cold-washing the carrier obtained in the previous step and then detecting the label bound to the carrier. Hepatitis test method. The method according to claim 1, wherein the lectin is Jacalin. The method according to claim 1 or 2, wherein the cold state is 1 to 10 ° C. The method according to any one of claims 1 to 3, wherein the enzyme-labeled second antibody is an HRP-labeled human IgM rabbit antibody, and the label is sensed by absorbance at 450 nm.

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